https://scholars.lib.ntu.edu.tw/handle/123456789/63802
標題: | 微米圖樣的製備及其生醫之應用 Microfabrication and biomedical applications of micropatterning |
作者: | 簡秀紋 Chien, Hsiu-Wen |
關鍵字: | 微米製程;表面化學;細胞-基材的相互作用;細胞-細胞的相互作用;共培養;micropatterning;surface chemistry;cell-substrate interaction;cell-cell interaction;co-culture | 公開日期: | 2012 | 摘要: | 生醫材料表面圖樣化可以用於許多基本的細胞生理研究,以及含細胞之生物感應器和組織工程的應用中。藉由表面的微米圖樣,控制細胞的形狀、細胞貼附的區域,以及細胞與細胞間或與基材表面之間的相互作用。該實驗的目的,藉由結合微製程的技術和表面修飾的方法,設計細胞貼附與抑制細胞貼附的微圖樣表面,並藉由細胞選擇性地貼附,形成細胞圖樣,使其應用在多種的生醫領域。 第一部分,本研究計畫希望開發出利用聚電解質多層膜結合光微影技術,發展出簡易的微圖樣表面的化學與地形特性的操控技術,控制細胞在微圖樣表面的貼附。將光交聯劑,芳香基疊氮化物,接於PAA上,成為可光交聯的聚電解質(PAA-Az)。將PAA-Az佈建在PAA/PAAm多層膜內,經由UV照射後,被曝曬到的區域會交聯,未曝曬的區域再使用鹼性的水洗去,就得到所要的圖形。我們發現,細胞只貼附在基材的區域,而不是PAA/PAAm多層膜的頂層。為了能更進一步控制細胞貼付的區域,依據細胞的貼附性,藉由交聯生物分子在基材或PAA/PAAm多層膜的頂層之表面,以加強細胞排列的趨勢,並進一步應用在肝細胞和非實質細胞的共培養系統。 第二部分,我們發展一個聚多巴胺的表面圖樣。藉由微接觸印刷法,先在聚二甲基矽氧烷的印章表面上沾上聚多巴胺,然後再轉印在幾種基材上,包括玻璃、矽晶片、聚苯乙烯和聚乙二醇等。這個黏著在基材上的聚多巴胺保有它獨特的反應性,藉由第二次反應,可調控微米圖樣的化學性質,產生多功能的表面。聚乙二醇基材上含有的聚多巴胺微米圖樣,可促進蛋白的吸附或細胞的貼附,形成蛋白圖樣或是細胞圖樣。另外,透過Michael 加成或是Schiff base反應,可將具有一級胺或是硫醇的分子與表面多巴胺反應,使分子接枝在表面。例如,原來促進細胞貼附的聚多巴胺表面,經過修飾一級胺含有的聚乙二醇後,便可轉換成抑制細胞貼附的表面。我們也發現到,奈米金顆粒可以直接地沉積在聚多巴胺的微圖樣,形成奈米金圖樣。除此之外,聚多巴胺也可應用在無電電鍍。當銀離子接觸到聚多巴胺,會自發性地與表面接合,使表面金屬化,析出奈米銀顆粒。結合微接觸印刷法與聚多巴胺,我們可以提供一個簡易又有經濟效益的方法,製造一個多功能的微米圖樣,藉由二次反應,可應用在多種領域上。 進一步地,我們延續第二部份的研究,結合聚多巴胺的化學特性,調控微米圖樣的表面化學,控制細胞在微圖樣表面的貼附。在此研究,我們摻混多巴胺與聚乙稀亞胺(PEI)的共聚物,例如PEI、PEI-g-PEG和PEI-g-galactose。以多巴胺與PEI共聚物的混合溶液作為墨汁,藉由微接觸印刷法,轉印在基材上。在原先不含PEI-g-PEG的聚多巴胺微米圖樣會促進L929細胞貼附;加入PEI-g-PEG後,微米圖樣會抑制細胞貼附。在原先不含PEI或PEI-g-galactose的聚多巴胺微米圖樣,PC12和HepG2/C3A細胞無法貼附在上;加入PEI後,促進PC12細胞貼附在圖樣上,而加入PEI-g-galactose後,促進HepG2/C3A細胞形成細胞圖樣,並應用在肝細胞和非實質細胞的共培養系統。藉由摻混PEI共聚物,我們提供一個簡易的方法,製造不同細胞種類的微米圖樣。 Cellular patterning on biomaterial surfaces is important in fundamental studies of cell–cell and cell–substrate interactions, and in biomedical applications such as tissue engineering, cell-based biosensors, and diagnostic devices. The objective of this study was to design selective adhesion of cells to a substrate with a region of adhesive surface and nonadhesive surface representing a pattern. The control cell spatial adhesion and the development of cellular pattering by incorporated microfabrications and surface modifications were applied to various biomedical fields. The first part, we combined the layer-by-layer polyelectrolyte multilayer deposition and photolithography to create an easy and versatile technique for cell patterning. Poly(acrylic acid) (PAA) conjugated with 4-azidoaniline was interwoven in PAA/polyacrylamide (PAM) multilayer films. After UV irradiation through a photo mask, the UV-exposed areas were crosslinked and the unexposed areas were rinsed away by alkaline water, resulting in micropatterns. Cell patterns were formed when the cell adhesion was limited to the base substrate, but not on the multilayer films. The stability of cell patterns could be modulated by simply modification of the surface chemistry of base substrate and PEM films with conjugation of bioactive macromolecules. Cell co-culture systems can be also achieved by this technique. The second part, a simple technique was developed to fabricate tuneable micropatterned substrates based on mussel-inspired surface modification. Polydopamine (PDA) was developed on polydimethylsiloxane (PDMS) stamps and was easily imprinted to several substrates such as glass, silicon, gold, polystyrene and polyethylene glycol via microcontact printing. The imprinted PDA retained its unique reactivity and could modulate the chemical properties of micropatterns via secondary reactions, which was illustrated in this study. PDA patterns imprinted onto a cytophobic and nonfouling substrates were used to form patterns of cells or proteins. PDA imprints reacted with nucleophilic amines or thiols to conjugate molecules such as polyethylene glycol for creating non-fouling area. Gold nanoparticles were immobilized onto PDA-stamped area. The reductive ability of PDA transformed silver ions to elemental metals as an electroless process of metallization. This facile and economic technique provides a powerful tool for development of a functional patterned substrate for various applications. Next, we modified a technique combining mussel-inspired surface chemistry and micro-contact printing (uCP) to modulate surface chemistry for cell patterning. The cell affinity of PDA was modulated by co-deposition with several poly(ethylene imine) (PEI)-based copolymers such as PEI, PEI-g-PEG (poly(ethylene glycol)) and PEI-g-galactose. The imprints of PDA/PEI-g-PEG benefit the formation of cell patterns on cell-favorable substrates. Neuronal PC12 cells were patterned via imprinting of PDA/PEI, while HepG2/C3A cells were arranged on the imprint of PDA/PEI-g-galactose. Finally, co-culture of HepG2/C3A cells and L929 fibroblasts was accomplished by our micro-patterning approach. This study demonstrated this facile and economic technique provides a powerful tool for development of functional patterned substrates for cell patterning. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/252127 |
顯示於: | 化學工程學系 |
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ntu-101-F95524036-1.pdf | 23.54 kB | Adobe PDF | 檢視/開啟 |
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